The synthesis of nucleotides in organisms is required for the cells in those organisms to divide and replicate. Nucleotide synthesis in mammals may be achieved through one of two pathways: the de novo synthesis pathway or the salvage pathway. Different cell types use these pathways to a different extent.
Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme involved in the de novo synthesis of guanosine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP) (Jackson R. C. et. al., Nature, 256:331-333 (1975)).
IMPDH is ubiquitous in eukaryotes, bacteria and protozoa (Y. Natsumeda & S. F. Carr, Ann. N.Y. Acad., 696:88-93 (1993)). The prokaryotic forms share 30-40% sequence identity with the human enzyme. Regardless of species, the enzyme follows an ordered Bi—Bi reaction sequence of substrate and cofactor binding and product release. First, IMP binds to IMPDH. This is followed by the binding of the cofactor NAD. The reduced cofactor, NADH, is then released from the product, followed by the product, XMP (S. F. Carr et al. J. Biol. Chem., 268:27286-90 (1993); E. W. Holmes et al. Biochim. Biophys. Acta, 364: 209-217 (1974)). This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require NAD to bind before the substrate.
Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced (F. R. Collart and E. Huberman J. Biol. Chem., 263:15769-15772 (1988); Y. Natsumeda et. al. J. Biol. Chem., 265:5292-5295 (1990)). Each is 514 amino acids, and they share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa (Y. Yamada et. al. Biochemistry, 27:2737-2745 (1988)).
The de novo synthesis of guanosine nucleotides, and thus the activity of IMPDH, is important in B and T-lymphocytes. These cells depend on the de novo, rather than salvage pathway to generate sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen (A. C. Allison et. al., Lancet II, 1179 (1975); A. C. Allison et. al. Ciba Found. Symp., 48:207 (1977)). Thus, IMPDH is an attractive target for selectively inhibiting the immune system without also inhibiting the proliferation of other cells.
Immunosuppression has been achieved by inhibiting a variety of enzymes including for example, the phosphatase calcineurin (inhibited by cyclosporin and FK-506); dihydroorotate dehydrogenase, an enzyme involved in the biosynthesis of pyrimidines (inhibited by leflunomide and brequinar); the kinase FRAP (inhibited by rapamycin); and the heat shock protein hsp70 (inhibited by deoxyspergualin). (See B. D. Kahan Immunological Reviews, 136:29-49 (1993); R. E. Morris The Journal of Heart and Lung Transplantation, 12(6):S275-S286 (1993)).
It is also known that IMPDH plays a role in other metabolic events. Increased IMPDH activity has been observed in rapidly proliferating human leukemic cell lines and other tumor cell lines, indicating IMPDH as a target for anti-cancer as well as immunosuppressive chemotherapy (M. Nagai et. al. Cancer Res., 51:3886-3890 (1991)). IMPDH has also been shown to play a role in the proliferation of smooth muscle cells, indicating that inhibitors of IMPDH, such as MPA or rapamycin, may be useful in preventing restenosis or other hyperproliferative vascular diseases (C. R. Gregory et al. Transplantation, 59:655-61 (1995); PCT publication WO 94/12184; and PCT publication WO 94/01105).
Additionally, IMPDH has been shown to play a role in viral replication in some viral cell lines. (S. F. Carr J. Biol. Chem., 268:27286-27290 (1993)). For instance, Merimepodib, an IMPDH inhibitor, has been proposed for the treatment of Hepatitis C Virus (HCV) infection. Analogous to lymphocyte and tumor cell lines, the implication is that the de novo, rather than the salvage, pathway is critical in the process of viral replication.
Inhibitors of IMPDH are also known. U.S. Pat. Nos. 5,380,879 and 5,444,072, and PCT publications WO 94/01105 and WO 94/12184 describe mycophenolic acid (MPA) and some of its derivatives as potent, uncompetitive, reversible inhibitors of human IMPDH type I (Ki=33 nM) and type 11 (Ki=9 nM). MPA has been demonstrated to block the response of B and T-cells to mitogen or antigen (A. C. Allison et. al. Ann. N.Y. Acad. Sci., 696:63 (1993)).
Immunosuppressants, such as MPA, are useful drugs in the treatment of transplant rejection and autoimmune diseases. (R. E. Morris Kidney Intl., 49:Suppl:53:S-26 (1996)). However, MPA is characterized by undesirable pharmacological properties, such as gastrointestinal toxicity and poor bioavailability. (L. M. Shaw, et. al., Therapeutic Drug Monitoring, 17:690-699 (1995)). Nucleoside analogs such as tiazofurin, ribavirin and mizoribine also inhibit IMPDH (L. Hedstrom, et. al. Biochemistry, 29:849-854 (1990)). These compounds, which are competitive inhibitors of IMPDH, suffer from lack of specificity to this enzyme.
Mycophenolate mofetil, a prodrug which quickly liberates free MPA in vivo, was recently approved to prevent acute renal allograft rejection following kidney transplantation. (L. M. Shaw, et. al., Therapeutic Drug Monitoring, 17:690-699 (1995); H. W. Sollinger Transplantation, 60:225-232 (1995)). Several clinical observations, however, limit the therapeutic potential of this drug. (L. M. Shaw, et. al., Therapeutic Drug Monitoring, 17:690-699 (1995)). MPA is rapidly metabolized to the inactive glucuronide in vivo. (A. C., Allison and E. M. Eugui Immunological Reviews, 136:5-28 (1993)). The glucuronide then undergoes enterohepatic recycling causing accumulation of MPA in the gastrointestinal tract where it cannot exert its IMPDH inhibitory activity on the immune system. This effectively lowers the drug's in vivo potency, while increasing its undesirable gastrointestinal side effects.
Numerous conditions, e.g., proliferative conditions such as leukemia and lymphoma, autoimmune diseases, viral infections, and the need for immunosuppression e.g., in the context of transplant therapy, cancer, inflammatory conditions such as rhinitis, inflammatory bowel disease, asthma, and several dermatological diseases can be treated with compounds that inhibit IMPDH. Although campounds that inhibit IMPDH are in use and have shown effectiveness, toxicity and other side effects have limited their usefulness.
Improving the delivery of drugs and other agents to target cells and tissues has been the focus of considerable research for many years. Though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory. Optimizing the association of the inhibitory drug with its intracellular target, while minimizing intercellular redistribution of the drug, e.g., to neighboring cells, is often difficult or inefficient.
Most agents currently administered to a patient parenterally are not targeted, resulting in systemic delivery of the agent to cells and tissues of the body where it is unnecessary, and often undesirable. This may result in adverse drug side effects, and often limits the dose of a drug (e.g., glucocorticoids and other anti-inflammatory drugs) that can be administered. By comparison, although oral administration of drugs is generally recognized as a convenient and economical method of administration, oral administration can result in either (a) uptake of the drug through the cellular and tissue barriers, e.g., blood/brain, epithelial, cell membrane, resulting in undesirable systemic distribution, or (b) temporary residence of the drug within the gastrointestinal tract. Accordingly, a major goal has been to develop methods for specifically targeting agents to cells and tissues. Benefits of such treatment includes avoiding the general physiological effects of inappropriate delivery of such agents to other cells and tissues, such as uninfected cells.
Thus, there is a need for therapeutic agents that inhibit IMPDH with improved pharmacological properties, e.g., drugs having improved IMPDH-inhibitory activity and pharmacokinetic properties, including improved oral bioavailability, greater potency and extended effective half-life in vivo. Such inhibitors would have therapeutic potential as immunosuppressants, anti-cancer agents, anti-vascular hyperproliferative agents and/or as anti-viral agents. Specifically, such compounds may be used in the treatment of transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease, as well as in the treatment of cancer and tumors, such as lymphomas and leukemia, vascular diseases, such as restenosis, and viral replication diseases, such as retroviral diseases and herpes. New IMPDH inhibitors should have fewer side effects, less complicated dosing schedules, and be orally active. In particular, there is a need for a less onerous dosage regimen, such as one pill, once per day.
Assay methods capable of determining the presence, absence or amounts of IMPDH inhibition, proliferation, immune response, and/or inflammation are of practical utility in the search for inhibitors as well as for diagnosing the presence of conditions associated with IMPDH activity.